Large-Scale Conformational Changes in Regulation of Transcription Initiation

描述（由申请人提供）：启动子DNA序列处的DNA依赖性RNA聚合酶（RNAP）的转录起始涉及由结合自由能驱动的大规模构象变化。对代表性大肠杆菌酶的这项研究的长期目标是表征这些构象变化的序列，并确定它们在创建转录泡和RNA合成的活性位点中的作用。在下一个项目期间，结合和解离的动力学研究将与低分辨率结构表征（化学和酶足迹法;荧光共振能量转移（FRET））一起进行，以确定每个步骤中发生的构象变化，以及每个步骤如何为下一步奠定基础。关键的具体目标包括阐明非特异性DNA序列从起始位点上游60-80个碱基对的包裹如何大大加速开放复合物的形成，并确定何时发生RNA聚合酶关键区域的重排（例如，从活性位点通道中弹出sigma 70掩蔽结构域，聚合酶非结构化区域的折叠）。为此，将表征野生型和缺失聚合酶突变体在缺失上游或下游序列的启动子变体处的开放复合物形成。显着的，但更简单的系统表现出类似的大规模的构象变化将进行平行研究：特异性结合的2个转录因子（乳糖阻遏物二聚体头，IHF）和非特异性结合的2个类核蛋白（HU，IHF）。温度和溶液变量对形成包裹的IHF-H' DNA界面的热力学的影响将通过等温滴定量热法（ITC）和FRET来测量，以获得和解释DNA包裹的热力学特征。将使用ITC确定多种结合模式和协同性在HU非特异性结合中的作用。耦合的局部折叠和形成的蛋白质接口的操作员结合将进行调查的交联二聚体的头部ITC，圆二色性和竞争性硝酸纤维素过滤测定与完整的紫胶阻遏物四聚体。对转录起始机制的理解进展影响人类健康的两个主要领域。首先，了解转录网络如何对发育和环境线索做出反应，对于识别和解决导致人类疾病（如癌症）的调控失败至关重要。其次，由细菌感染引起的人类疾病（例如肺结核）对公共卫生造成严重破坏。该提议产生的结果将定义细菌特异性的启动方面，从而揭示下一代抗生素的新靶点。

DESCRIPTION (provided by applicant): Transcription initiation by DNA-dependent RNA polymerases (RNAP) at promoter DNA sequences involves large-scale conformational changes driven by binding free energy. The long term goal of this research on the representative E, coli enzyme is to characterize the sequence of these conformational changes and to determine their roles in creating the transcription bubble and active site for RNA synthesis. In the next project period, kinetic studies of association and dissociation will be performed in concert with low resolution structural characterizations (chemical and enzymatic footprinting; fluorescence resonance energy transfer (FRET)) to determine what conformational changes occur in each step, and how each sets the stage for the next. Key specific aims include elucidating how wrapping of nonspecific DNA sequences 60-80 base pairs upstream from the start site greatly accelerates open complex formation, and identifying when rearrangements in key regions of RNA polymerase (e.g. ejection of sigma70 masking domain from the active site channel, folding of unstructured regions of polymerase) occur. To this end, open complex formation by wild type and deletion polymerase mutants at promoter variants that delete upstream or downstream sequences will be characterized. Significant but simpler systems exhibiting analogous large-scale conformational changes will be studied in parallel: specific binding of 2 transcription factors (lac repressor dimer headpiece, IHF); and nonspecific binding of 2 nucleoid proteins (HU, IHF). Effects of temperature and solution variables on the thermodynamics of formation of a wrapped IHF-H' DNA interface will be measured by isothermal titration calorimetry (ITC) and FRET to obtain and interpret the thermodynamic signatures of DNA wrapping. Roles of multiple binding modes and cooperativity in nonspecific binding of HU will be determined using ITC. Coupling of local folding and formation of protein interfaces to operator binding will be investigated for the crosslinked dimeric headpiece by ITC, circular dichroism and a competitive nitrocellulose filter assay with intact lac repressor tetramer. Advances in the understanding of the mechanism of transcription initiation impacts 2 main areas of human health. First, understanding how transcriptional networks respond to developmental and environmental cues is fundamental to identifying and addressing failures in regulation that cause human diseases such as cancer. Secondly, human illnesses caused by bacterial infection (e.g. tuberculosis) wreak havoc on public health. Results generated by this proposal will define bacteria-specific aspects of initiation, and thus, reveal novel targets for the next generation of antibiotics.